A central problem for the development of HIV-1 vaccines has been to identify immunogen determinant capable of raising high titer, long lasting, neutralizing antibody against the envelope glycoproteins (Envs) of primary isolates. The HIV-1 Env has proven to be a weak immunogen raising low titer antibodies that are slow to undergo affinity maturation. In this proposal, DNA and protein immunogens will be used to test the C3d component of complement as a molecular adjuvant for Env. In normal immune responses the attachment of C3d to an antigen enhances both the initiation and the maturation of antigen-specific antibody. To test a potential adjuvant role of C3d for Env, DNA constructs will be produced encoding Env fused to C3d. These constructs will be used for DNA immunizations and to produce protein for protein immunizations. The study will be executed according to three specific aims. (l) Vaccinate with plasmids encoding secreted monomeric (gpl20) and oligomeric (gpl40) forms of a primary Env (89.6) and using these same forms of Env fused at the carboxyl terminus to one, two or three tandem copies of C3d will be constructed. (2) Plasmid constructs will be expressed to (a) determine the levels of expression and secretion of the various Envs, (b) to test the various Envs for the maintenance of native/functional conformations, and (c) to purify selected Envs for protein immunizations. (3) Env constructs will be compared for immunogenicity in mice, using DNA immunizations. For the more favorable constructs, DNA immunizations plus protein boosts, and protein immunizations will be conducted. Raised antibody will be titered for the level of Env-specific IgG and for neutralizing activity for a panel of primary as well as T-cell line adapted HIV-1 isolates. Our goal is to identify the most favorable Env-C3d fusion for raising high titer neutralizing antibody for HIV-l. The hypothesis throughout the study is that the C3d fusions will increase the immunogenicity of Env both by increasing the efficiency of the initiation of anti-Env Ab responses and by increasing the ability of anti-Env Ab to undergo affinity maturation.